A novel carboxymethylcellulose–gelatin–titanium dioxide–superoxide dismutase biosensor; electrochemical properties of carboxymethylcellulose–gelatin–titanium dioxide–superoxide dismutase Emel Emregul ⁎, Ozge Kocabay, Burak Derkus, Tugrul Yumak, Kaan Cebesoy Emregul, Ali Sınag, Kamran Polat Ankara University, Science Faculty, Department of Chemistry, Tandoğan, Ankara, 06100, Turkey abstract article info Article history: Received 4 July 2012 Received in revised form 3 September 2012 Accepted 17 September 2012 Available online 3 October 2012 Keywords: Carboxymethylcellulose–gelatin TiO 2 nanoparticles Superoxide dismutase Impedance spectroscopy Biosensor A novel highly sensitive electrochemical carboxymethylcellulose–gelatin–TiO 2 –superoxide dismutase biosensor for the determination of O 2 •- was developed. The biosensor exhibits high analytical performance with a wide linear range (1.5 nM to 2 mM), low detection limit (1.5 nM), high sensitivity and low response time (1.8 s). The electron transfer of superoxide dismutase was first accomplished at the carboxymethylcellulose–gelatin– Pt and carboxymethylcellulose–gelatin–TiO 2 –Pt surface. The electron transfer between superoxide dismutase and the carboxymethylcellulose–gelatin–Pt wihout Fe(CN) 6 4-/3- and carboxymethylcellulose–gelatin–Pt, car- boxymethylcellulose–gelatin–TiO 2 –Pt with Fe(CN) 6 4-/3- is quasireversible with a formal potential of 200 mV, 207 mV, and 200 mV vs Ag|AgCl respectively. The anodic (ks a ) and cathodic (ks c ) electron transfer rate constants and the anodic (α a ) and cathodic (α c ) transfer coefficients were evaluated: ks a =6.15 s -1 , α a =0.79, and ks c = 1.48 s -1 α c =0.19 for carboxymethylcellulose–superoxide dismutase without Fe(CN) 6 4-/3- , ks a =6.77 s -1 , α a =0.87, and ks c =1 s -1 α c =0.13 for carboxymethylcellulose–superoxide dismutase with Fe(CN) 6 4-/3- , ks a =6.85 s -1 , α a = 0.88, and ks c =0.76 s -1 α c =0.1 carboxymethylcellulose–gelatin–TiO 2 –superoxide dismutase. The electron transfer rate between superoxide dismutase and the Pt electrode is remarkably en- hanced due to immobilizing superoxide dismutase in carboxymethylcellulose–gelatin and TiO 2 nanoparticles tend to act like nanoscale electrodes. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Superoxide dismutases (SOD) which play an important role in cell protection mechanisms against oxidative damage from reactive oxygen species are ubiquitous metalloenzymes in oxygen-tolerant organisms [1–3]. Oxygen radicals has attracted considerable attention due to their harmful interaction with biological molecules and their involvement in signaling pathways. Superoxide radical (O 2 •- ), the primary species of the reactive oxygen species (ROS), plays a central role in physiological processes [4–6]. Under normal metabolic conditions, O 2 •- is produced at a rate that is matched by the capacity of tissue to catabolize them [7]. When its production exceeds the body's natural ability to deal with the potentially cytotoxic species, a variety of pathological condi- tions may result including cancer, stroke, and neurodegeneration [8]. In plants, O 2 •- is commonly produced in illuminated chloroplasts by the occasional transfer of an electron from an excited Chl molecule or PSI components under conditions of high NADPH/NADP ratios to molecular O 2 [9]. Various environmental perturbations, such as hyperoxia, herbicides, pathogens, ozone, temperature fluctuations, and other stresses are known to induce O 2 •- formation in most aerobic organisms [9]. In order to understand the role of O 2 •- in pathology and physiology and the relationship between O 2 •- and environmental stresses, it is essential to determine O 2 •- in a variety of in vitro and in vivo models. Therefore, the quantitative determination of O 2 •- concentrations and the beneficial effects of antioxidant compounds is of great interest to the medical community. Due to its low con- centration, high reactivity, and short lifetime, it is still an analytical challenge to detect the local concentration of O 2 •- , especially in the biological systems. Determination of free radicals is usually carried out with spectrometry, fluorometry, chemiluminensence, and elec- tron spin resonance [10–18]. Electron transfer reactions from redox enzymes, to electrodes have been widely studied, due to their signif- icant assignments in physiological reactions, biotechnology and also in the development of biosensors and bioelectronic devices [19–23]. Protein electrochemistry has been shown to allow real-time, on-line quantification of radical concentrations. Recent attempts have con- centrated on electrochemical methods due to their direct, real-time measurements and capability for in vivo detection [24–27]. Mostly, copper, zinc-superoxide dismutase (Cu, Zn–SOD)-immobilized elec- trodes have paved an elegant way to detect O 2 •- . SODs catalyze the dismutation of O 2 •- to O 2 and H 2 O 2 via a cyclic oxidation -reduction electron-transfer mechanism and are widely distributed among aero- bic organisms and show high rate constants, up to the order of Bioelectrochemistry 90 (2013) 8–17 ⁎ Corresponding author. E-mail address: eemregul@yahoo.com (E. Emregul). 1567-5394/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bioelechem.2012.09.002 Contents lists available at SciVerse ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem